Rhea Mehta

Fructose and carbonyl metabolites as endogenous toxins

Dietary fructose consumption is one of the environmental factors contributing to the development of obesity and a fatty liver (hepatic steatosis). A two-hit hypothesis has been proposed for progression of hepatic steatosis to the more serious non-alcoholic steatosis (NASH), with the first hit being hepatic steatosis, and the second hit being inflammation and associated oxidative stress caused by reactive oxygen species (ROS) formation. As well, fructose-fed rats develop insulin resistance and serum levels of methylglyoxal, a glycolytic metabolite, are increased. Previously we reported that glyoxal-induced hepatocyte cytotoxicity could be attributed to mitochondrial toxicity as mitochondrial membrane potential was decreased and cytotoxicity was increased several orders of magnitude by low non-cytotoxic doses of H(2)O(2) (hepatocyte inflammation model). In this study, we have assessed the toxicity of fructose towards hepatocytes and investigated the molecular cytotoxic mechanisms involved. Fructose itself was only toxic at 1.5M, whereas 12 mM caused 50% cell death in 2h if the hepatocytes were exposed to a non-cytotoxic dose of H(2)O(2) continuously generated by glucose and glucose oxidase. The cytotoxic mechanism involved oxidative stress as ROS and H(2)O(2) formation preceded cytotoxicity, and cytotoxicity was prevented by radical scavengers, lipid antioxidants and ROS scavengers. It is proposed that the highly potent Fenton derived ROS catalyse the oxidation of fructose and particularly its carbonyl metabolites glycolaldehyde, dihydroxyacetone, glyceraldehyde. The carbon radicals and glyoxal formed compromise the cells resistance to H(2)O(2).

Cytoprotective mechanisms of carbonyl scavenging drugs in isolated rat hepatocytes

Diabetes is a disease among several others that has been linked with the accumulation of carbonylated proteins in tissues. Carbonylation is an irreversible, non-enzymatic modification of proteins by carbonyls. In Diabetes, dicarbonyls are thought to be generated by the autoxidation of reducing sugars which react with proteins and eventually lead to the formation of advanced glycation end-products (AGEs). Carbonyl scavenging drugs containing thiol or amine functional groups have been suggested to act therapeutically in preventing protein carbonylation by trapping the dicarbonyls glyoxal and methylglyoxal to form non-toxic adducts. This study seeks to determine the mechanism by which carbonyl scavenging drugs prevent glyoxal toxicity in a cell-free system as well as in isolated rat hepatocytes. In a cell free system, the glyoxal trapping ability of the drugs was measured by following glyoxal disappearance using Girards Reagent T. For the thiol-containing drugs, the order of effectiveness for glyoxal trapping was penicillamine>cysteine>N-acetyl-cysteine, whereas for the amine-containing drugs, the order of effectiveness for glyoxal trapping was aminoguanidine>>pyridoxamine>metformin. Furthermore, aminoguanidine, penicillamine and cysteine at concentrations equimolar to glyoxal prevented protein carbonylation. Other scavenging drugs such as pyridoxamine, hydralazine or metformin did not prevent glyoxal-induced cytotoxicity by trapping glyoxal, but instead prevented cytotoxicity by acting as a mitochondrial vitamin, lipid antioxidant or reactive oxygen species scavenger, respectively.

Differences in glyoxal and methylglyoxal metabolism determine cellular susceptibility to protein carbonylation and cytotoxicity

Chronic hyperglycemia in diabetic patients often leads to chronic side effects associated with protein glycation and the formation of reactive carbonyl species, such as methylglyoxal (MGO) and glyoxal (GO). We have shown that both MGO and GO carbonylated bovine serum albumin (BSA) in vitro to the same degree and stability. The carbonylated BSA formed initially could be a reversible Schiff base as the UV absorbance formed after the addition of 2,4-dinitrophenylhydrazine was decreased when sodium borohydride was added. MGO and GO also carbonylated hepatocyte protein rapidly with similar dose and time dependence. In contrast to BSA carbonylation, the amount of carbonylated proteins in hepatocytes decreased over time, much more rapidly for hepatocytes treated with MGO than with GO. This could be attributed to the rapid hepatocyte metabolism of MGO with glyoxalase I, the predominant detoxification enzyme for MGO. Protein carbonylation and the associated toxicity caused by GO and MGO were studied in the following hepatocyte models: (1) control hepatocytes, (2) glutathione (GSH)-depleted hepatocytes, (3) mitochondrial aldehyde dehydrogenase (ALDH2)-inhibited hepatocytes, (4) hepatocyte inflammation model, and (5) catalase-inhibited hepatocyte model. Carbonylation and cytotoxicity caused by MGO or GO was markedly increased in GSH-depleted hepatocytes as compared to control hepatocytes. Hepatocytes exposed to non-toxic concentrations of H(2)O(2) or hepatocytes treated with catalase inhibitors also showed a marked increase in GO-caused cytotoxicity and protein carbonylation, whereas there were only minor increases with MGO. The GO effect was attributed to potential radical formation and the inhibition effect of H(2)O(2) on aldehyde dehydrogenase, a major GO metabolising enzyme. GO-caused cytotoxicity and protein carbonylation were also increased with ALDH2-inhibited hepatocytes whereas such an increase was only observed with MGO in GSH-depleted hepatocytes.

Hepatocyte inflammation model for cytotoxicity research: fructose or glycolaldehyde as a source of endogenous toxins

Insulin resistance and hepatotoxicity induced in high fructose fed rats may involve fructose derived endogenous toxins formed by inflammation. Thus fructose was seventy-fold more toxic if hepatocytes were exposed to non-toxic levels of hydrogen peroxide (H2O2) released by inflammatory cells. This was prevented by iron (Fe) chelators, hydroxyl radical scavengers, and increased by Fe, copper (Cu) or catalase inhibition. Fructose or glyceraldehyde/dihydroxyacetone metabolites were oxidized by Fenton radicals to glyoxal. Glyoxal (15μM) cytotoxicity was increased about 200-fold by H2O2. Glycolaldehyde was enzymically formed from glyceraldehyde, the fructokinase/aldolase B product of fructose. Glycolaldehyde cytotoxicity was increased 20-fold by H2O2. The oxidative stress cytotoxicity induced was attributed to the Fenton oxidation of glycolaldehyde forming glycolaldehyde radicals and glyoxal, since cytotoxicity was prevented by aminoguanidine (glyoxal trap) or Fenton inhibitors. Glyoxal was also the Fenton product responsible for glycolaldehyde protein carbonylation as carbonylation was prevented by aminoguanidine or Fenton inhibitors.

Risk factors for colorectal cancer in man induce aberrant crypt foci in rats: Preliminary findings

ABSTRACT Epidemiological studies have demonstrated clear associations between specific dietary and environmental risk factors and incidence of colorectal cancer, but the mechanisms responsible for these associations are not known. An animal model could facilitate such an understanding. Both genotoxic and nongenotoxic carcinogens induce aberrant crypt foci (ACF) in the colons of F344 rats. F344 rats were provided with diets that contained putative risk factors for CRC: low calcium and low vitamin D, high iron, high fructose, and decreased light (UV) exposure or a control diet for 14 wk. The rats were then assessed with biochemical measures and by topological examination for evidence of colon abnormalities. Circulating ionized calcium was decreased from 2.85 to 1.69 mmol/L, and ACF were increased from 0.7 to 13.6 lesions/colon (both P < 0.001). Rats exposed to the multiple environmental conditions associated with colon cancer, developed ACF similar to the heterogeneous or ill-defined ACF in the human colon. Heterogeneous ACF are the most frequently seen in humans and are also seen in rats shortly after exposure to the non-genotoxic colon carcinogen, dextransulfate sodium. The rodent model could be used to assess the pathways from diet and environment to colon cancer and to provide guidance for clinical studies.